Kinase Activity Of DNA-dependent Protein Kinase Research Articles

Abstract A001: Discovery of imidazo [4,5-c]pyridin-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitizers

Abstract Inhibition of the repair of radiation-induced DNA double strand breaks (DSB) offers the potential to sensitize tumors to radiation therapy. The dominant role of non-homologous end-joining in the repair of radiation-induced DSBs indicates that DNA-dependent protein kinase (DNA-PK) is an excellent target for the development of radiosensitizers. We report the discovery of a new chemical class of kinase inhibitor based on the imidazo[4,5-c]pyridin-2-one scaffold. The class was developed by scaffold-hopping from the pan phosphoinositide 3-kinsase (PI3K) and PI3K-like kinase (PIKK) inhibitor dactolisib. Iterative development culminated in the identification of SN39536 as a low nM DNA-PK inhibitor with excellent selectivity. Further modification led to the discovery of the more potent and more selective SN40905. Both SN39536 and SN40905 were effective inhibitors of DNA-PK kinase activity in a biochemical assay with substantial selectivity for DNA-PK across the PIKK and PI3K families. Both compounds selectively inhibited growth of HAP1 PRKDC wild-type cells when combined with radiation, but not the corresponding PRKDC-/- cells. SN39536 and SN40905 were effective radiosensitizers of colorectal carcinoma (HCT116), non-small cell lung cancer (H460, H1299, and A549), pancreatic (BxPC-3, PANC-1, and MiaPaCa-2) and head and neck squamous cell carcinoma (FaDu and UT-SCC-74B) cells determined using a clonogenic survival endpoint. Both SN39536 and SN40905 displayed high oral bioavailability. When administered PO at a range of non-toxic doses, both SN39536 and SN40905 provided significant additional tumor cell killing of HCT116, UT-SCC-74B, and BxPC-3 tumor xenografts in combination with a single (13 Gy) radiation treatment determined by ex vivo clonogenic survival assays. SN39536 also provided substantial tumor growth inhibition of HCT116 tumor xenografts in combination with RAD (10 Gy). SN39536 and SN40905 represent a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for the treatment of human cancers. Citation Format: Michael P. Hay, Cho R. Hong, Lydia P. Liew, Benjamin D. Dickson, Way W. Wong, Sophia F. O'Brien-Gortner, Rebecca Airey, Jagdish Jaiswal, William R. Wilson, Stephen M. Jamieson. Discovery of imidazo [4,5-c]pyridin-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitizers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A001.

Abstract B068: Modulation of the DNA damage response by novel Ku-DNA binding inhibitors enhances cellular effects of DNA-double strand break inducing agents

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is being pursued as a therapeutic strategy for the treatment of cancer in combination with DNA-double strand break inducing agents. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via a novel mechanism of action to inhibit DNA-PK catalytic kinase activity by targeting the Ku 70/80 heterodimer interaction with DNA. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity, and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating therapies bleomycin, etoposide and ionizing radiation. In this study, we have expanded our structure activity relationship analyses to focus on optimizing cellular uptake and reducing protein binding. We present the synthesis and analysis of a new series of Ku-DBi’s which demonstrated that chemical modifications of the X80 core structure can be implemented to achieve these goals while retaining Ku inhibitory activity. Furthermore, novel relationships with in vitro inhibitory activity were identified as a function of new pharmacophore additions. In addition, these novel compounds display reduced non-specific protein binding, increased cellular uptake, and enhanced cellular inhibition on DNA-double strand break repair. These novel compounds enable cellular studies to be conducted in serum contain media and the pursuit of in vivo analysis and represent a significant advance in the development of Ku-DNA binding inhibitors. Citation Format: Pamela L Mendoza-Munoz, Dineshsinha Chauhan, Navnath S Gavande, John J Turchi. Modulation of the DNA damage response by novel Ku-DNA binding inhibitors enhances cellular effects of DNA-double strand break inducing agents [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B068.

281 (PB061) - Ku-DNA binding inhibitors modulate the DNA damage response in response to DNA double-strand breaks

Background: The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is being pursued as a therapeutic strategy for the treatment of cancer in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory previously reported the development of Ku-DNA binding inhibitors (Ku-DBis) that act via inhibition of DNA-PK catalytic kinase activity by blocking the Ku-DNA interaction.

Abstract 2596: Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of bleomycin and IR and also induce p53 phosphorylation through the activation of the ATM pathway, which is concomitant with a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 and A549 cell lines, Ku-DBi’s treatment in combination with DNA DSBs-inducing agents such as bleomycin and IR, showed a significant reduction of autophosphorylation events of DNA-PKcs at the S2056 cluster compared to DNA DSBs-inducing agent alone. In addition, analysis of phospho-ATM and phospho-p53 protein levels in these NSCLC cell lines, suggested activation of the ATM pathway as a function of Ku-DBi’s treatment followed by bleomycin, evidenced by an increase of phosphorylation of ATM at Ser1981, and a modest increase of p53 phosphorylation at Ser15. Our findings demonstrate that Ku-DBi’s block DNA-DSB dependent DNA-PKcs autophosphorylation, resulting in potentiating cellular sensitivity to bleomycin and IR, and a likely effect on the ATM-dependent signaling pathway. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs to impact DSB repair and potentially DDR signaling, becoming in a promising approach as part of an anticancer therapeutic strategy in combination with DNA DSBs-inducing agents. Citation Format: Pamela L. Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2596.

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Abstract PO-023: Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double stand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a series of a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We recently reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of IR. We demonstrate that the increase in IR sensitivity is preceded by a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 cell line, Ku-DBi 245 treatment in combination with IR showed a significant reduction of phosphorylation of DNA-PKcs at S2056 compared to IR alone. Analysis of gH2A.X also showed an inhibition of the DDR as a function of 245 treatment, evidenced by reduction of S139 phosphorylation. These results demonstrate that Ku-DBi 245 blocks DNA-break dependent DNA-PKcs autophosphorylation, resulting in an increased in radiation sensitivity. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs and DDR signaling as part of an anticancer therapeutic strategy in combination with IR. Citation Format: Pamela Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy Garrett, John J. Turchi. Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-023.

Phosphoproteomics Uncovers Synergy between DNA-PK and FLT3 Inhibitors in Acute Myeloid Leukaemia

*These authors contributed equally to this work Background:Acute Myeloid Leukaemia (AML) is the most common and aggressive form of acute leukaemia, with a 5-year survival rate of just 24%. Activating mutations in the receptor tyrosine kinase FLT3 are the most common driver mutations in AML (25-30% of patients). Inhibiting the FLT3 receptor as a mono-therapeutic strategy in AML has proven difficult however, due to the development of treatment resistance and relapse. In order to identify improved therapeutic targets, the oncogenic signalling pathways downstream of mutant FLT3 require characterisation. Methods:Quantitative, label-based phosphoproteomics was performed on primary blasts from 7 AML patients (4 mutant-FLT3, 3 wildtype-FLT3). Differentially phosphorylated pathways were identified using Ingenuity Pathway Analysis, and kinase activation was assessed by kinase substrate enrichment analysis. Validation of results was performed using targeted mass spectrometry. Proliferation, apoptosis, and cell cycle assays were used to assess drug toxicity; drug synergy was evaluated using Chou-Talalay and Webb analyses. Results:Analysis of differentially expressed phosphoproteins in mutant-FLT3 compared to wildtype-FLT3 AML patient blasts revealed dysregulation of DNA repair pathways. Specifically, mutant-FLT3 samples displayed increased phosphorylation of proteins within the error-prone Non-Homologous End Joining (NHEJ) repair pathway, indicating NHEJ pathway activation. Kinase enrichment analysis predicted increased activity of the NHEJ core kinase, DNA-dependent protein kinase (DNA-PK), in mutant-FLT3 samples. Accordingly, proliferation assays revealed that mutant-FLT3 cell lines were sensitive to inhibition of DNA-PK. FLT3-inhibitor treatment reduced DNA-PK phosphorylation in mutant-FLT3 cells, suggesting that activation of DNA-PK is downstream of FLT3 activation. Inhibition of DNA-PK kinase activity combined with FLT3 inhibitors led to synergistic induction of cell death, selectively in mutant-FLT3 cell lines. DNA-PK inhibitors combined with FLT3 inhibitors also co-operatively induced cell death in mutant-FLT3 primary AML patient samplesex vivo, and significantly prolonged survival compared to either monotherapy in a human AML xenograft mouse model. Conclusions:Mutant-FLT3 AML is associated with activation of the error-prone NHEJ repair pathway, which may contribute to genomic instability. Targeting the NHEJ kinase, DNA-PK, in combination with FLT3 inhibitors has the potential to improve outcomes for this poor-prognosis AML subtype. Disclosures Enjeti: Bayer:Speakers Bureau;AbbVie:Membership on an entity's Board of Directors or advisory committees;Alexion:Speakers Bureau;Novartis:Membership on an entity's Board of Directors or advisory committees;Astellas:Membership on an entity's Board of Directors or advisory committees;Sanofi:Speakers Bureau.

Phosphorylation Targets of DNA-PK and Their Role in HIV-1 Replication.

The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer. The kinase activity of DNA-PK is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). We also showed that the kinase activity of DNA-PK is essential for post-integrational DNA repair in the case of HIV-1 infection. Besides, DNA-PK is known to participate in such cellular processes as protection of mammalian telomeres, transcription, and some others where the need for its phosphorylating activity is not clearly elucidated. We carried out a systematic search and analysis of DNA-PK targets described in the literature and identified 67 unique DNA-PK targets phosphorylated in response to various in vitro and/or in vivo stimuli. A functional enrichment analysis of DNA-PK targets and determination of protein–protein associations among them were performed. For 27 proteins from these 67 DNA-PK targets, their participation in the HIV-1 life cycle was demonstrated. This information may be useful for studying the functioning of DNA-PK in various cellular processes, as well as in various stages of HIV-1 replication.

Sodium salicylate acts through direct inhibition of phosphoinositide 3-kinase-like kinases to modulate topoisomerase-mediated DNA damage responses

Chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs) exhibit diverse pharmacological and biological activities mainly through their inhibitory effect on cyclooxygenase (COX). However, COX-independent mechanisms involving kinase inhibition have been proposed to explain certain therapeutic effects of NSAIDs. Here, we explored the potential relationship between chemopreventive NSAIDs and DNA damage responses induced by treatment with topoisomerase-targeting drugs. (1) Sodium salicylate, a non-COX-selective NSAID, was shown to reduce DNA damage-induced RPA and p53 phosphorylation. (2) The formation of enzyme cleavable complexes by topoisomerase-targeting drugs was not affected in the presence of sodium salicylate. (3) The attenuating effect of NSAIDs on the DNA damage responses is COX-2-independent, since COX-2-selective inhibitors failed to inhibit DNA damage-induced phosphorylation of replication protein A (RPA) and p53. (4) This COX-2-independent attenuating effect was mediated through interference of neither nuclear factor kappa B nor extracellular signal-regulated kinase pathways. (5) The activation of ataxia telangiectasia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK), two key signal transducers upstream of RPA and p53, was found to be significantly reduced with sodium salicylate treatment. (6) Most importantly, sodium salicylate and other NSAIDs directly inhibited kinase activity of ATM and DNA-PK. The extent of inhibition on the kinase activity also correlated with the degree of attenuation on the DNA damage responses. (7) Unexpectedly, sodium salicylate showed a p53-independent protection effect on topoisomerase-mediated cell killing. Together, our study provides evidence that NSAIDs exhibit a novel COX-independent modulating activity of NSAIDs on the DNA damage responses and it is through inhibition of phosphoinositide 3-kinase-like kinases.

The human telomerase RNA component, hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1

Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres.

Aggregative organization enhances the DNA end‐joining process that is mediated by DNA‐dependent protein kinase

The occurrence of DNA double-strand breaks in the nucleus provokes in its structural organization a large-scale alteration whose molecular basis is still mostly unclear. Here, we show that double-strand breaks trigger preferential assembly of nucleoproteins in human cellular fractions and that they mediate the separation of large protein-DNA aggregates from aqueous solution. The interaction among the aggregative nucleoproteins presents a dynamic condition that allows the effective interaction of nucleoproteins with external molecules like free ATP and facilitates intrinsic DNA end-joining activity. This aggregative organization is functionally coacervate-like. The key component is DNA-dependent protein kinase (DNA-PK), which can be characterized as a DNA-specific aggregation factor as well as a nuclear scaffold/matrix-interactive factor. In the context of aggregation, the kinase activity of DNA-PK is essential for efficient DNA end-joining. The massive and functional concentration of nucleoproteins on DNA in vitro may represent a possible status of nuclear dynamics in vivo, which probably includes the DNA-PK-dependent response to multiple double-strand breaks.

Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)- t-butyl-4-hydroxyanisole or N-acetyl- l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PK pThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine ( SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PK pThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.

Phosphorylation of linker histones by DNA-dependent protein kinase is required for DNA ligase IV-dependent ligation in the presence of histone H1

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.

Enhanced DNA-dependent protein kinase activity in Sjögren's syndrome B cells.

To examine the stress response, including the role of DNA-dependent protein kinase (DNA-PK), in B cells from Sjögren's syndrome (SS) patients. B-cell lines were exposed to gamma radiation and then postincubated to allow inducible stress functions to develop. The magnitude of the DNA damage response was monitored with respect to DNA-PK phosphorylation of a p53 peptide, defence protein levels (Ku, DNA-PK catalytic subunit, ATM, p21 and p53) and flow cytometric determination of cell cycle phases and apoptosis. B cells from SS patients, compared with healthy controls, displayed enhancement of two stress functions in undamaged cells: DNA-PK kinase activity and apoptosis. In addition, SS showed enhanced cell cycle arrest in gamma-irradiated cells. Strong kinase activity of DNA-PK, functioning not only in a DNA damage response but also in immunoglobulin gene rearrangement, may be an important component of the heightened stress response displayed by SS cells. In combination with recent reports, our data indicate that constitutional hyper-reactivity to danger signals is a basic pathogenetic factor in SS.

A Positive Role for the Ku Complex in DNA Replication Following Strand Break Damage in Mammals

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.

The kinase activity of DNA-PK is required to protect mammalian telomeres

The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.

DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination

Nonhomologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. Several proteins, including the DNA-dependent protein kinase (DNA-PK), XRCC4 and DNA ligase IV, are required for nonhomologous end joining both in vitro and in vivo. Since XRCC4 is recruited to the DNA double-strand break with DNA-PK, and because the protein kinase activity of DNA-PK is required for its in vivo function, we reasoned that XRCC4 could be a potential physiological substrate of DNA-PK. Here, we have used mass spectrometry to map the DNA-PK phosphorylation sites in XRCC4. Two major phosphorylation sites (serines 260 and 318), as well as several minor sites were identified. All of the identified sites lie within the carboxy-terminal 100 amino acids of XRCC4. Substitution of each of these sites to alanine (in combination) reduced the ability of DNA-PK to phosphorylate XRCC4 in vitro by at least two orders of magnitude. However, XRCC4-deficient cells that were complemented with XRCC4 lacking DNA-PK phosphorylation sites were analogous to wild type XRCC4 with respect to survival after ionizing radiation and ability to repair DSBs introduced during V(D)J recombination.

Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

Protein Phosphatases Regulate DNA-dependent Protein Kinase Activity

DNA-dependent protein kinase (DNA-PK) is a complex of DNA-PK catalytic subunit (DNA-PKcs) and the DNA end-binding Ku70/Ku80 heterodimer. DNA-PK is required for DNA double strand break repair by the process of nonhomologous end joining. Nonhomologous end joining is a major mechanism for the repair of DNA double strand breaks in mammalian cells. As such, DNA-PK plays essential roles in the cellular response to ionizing radiation and in V(D)J recombination. In vitro, DNA-PK undergoes phosphorylation of all three protein subunits (DNA-PK catalytic subunit, Ku70 and Ku80) and phosphorylation correlates with inactivation of the serine/threonine protein kinase activity of DNA-PK. Here we show that phosphorylation-induced loss of the protein kinase activity of DNA-PK is restored by the addition of the purified catalytic subunit of either protein phosphatase 1 or protein phosphatase 2A (PP2A) and that this reactivation is blocked by the potent protein phosphatase inhibitor, microcystin. We also show that treating human lymphoblastoid cells with either okadaic acid or fostriecin, at PP2A-selective concentrations, causes a 50-60% decrease in DNA-PK protein kinase activity, although the protein phosphatase 1 activity in these cells was unaffected. In vivo phosphorylation of DNA-PKcs, Ku70, and Ku80 was observed when cells were labeled with [(32)P]inorganic phosphate in the presence of the protein phosphatase inhibitor, okadaic acid. Together, our data suggest that reversible protein phosphorylation is an important mechanism for the regulation of DNA-PK protein kinase activity and that the protein phosphatase responsible for reactivation in vivo is a PP2A-like enzyme.

Regulation of DNA-dependent Protein Kinase Activity by Ionizing Radiation-activated Abl Kinase Is an ATM-dependent Process

Ionizing radiation (IR) treatment results in activation of the nonreceptor tyrosine kinase c-Abl because of phosphorylation by ATM. In vitro evidence indicates that DNA-dependent protein kinase (DNA-PK) can also phosphorylate and thus potentially activate Abl kinase activity in response to IR exposure. To unravel the role of ATM and DNA-PK in the activation of Abl, we assayed Abl, ATM, and DNA-PK activity in ATM- and DNA-PKcs-deficient cells after irradiation. Our results show that despite the presence of higher than normal levels of DNA-PK kinase activity, c-Abl fails to become activated after IR exposure in ATM-deficient cells. Conversely, normal activation of both ATM and c-Abl occurs in DNA-PKcs-deficient cells, indicating that ATM but not DNA-PK is required for activation of Abl in response to IR treatment. Moreover, activation of Abl kinase activity by IR correlates well with activation of ATM activity in all phases of the cell cycle. These results indicate that ATM is primarily responsible for activation of Abl in response to IR exposure in a cell cycle-independent fashion. Examination of DNA-PK activity in response to IR treatment in Abl-deficient cells expressing mutant forms of Abl or in normal cells exposed to an inhibitor of Abl suggests an in vivo role for Abl in the down-regulation of DNA-PK activity. Collectively, these results suggest a convergence of the ATM and DNA-PK pathways in the cellular response to IR through c-Abl kinase.